The present invention relates to a method for screening substances that inhibit binding between TAK1 and TAB1. The present invention also relates to a method for screening substances that inhibit the signal transduction of transforming growth factor-xcex2 (TGF-xcex2). The present invention further relates to substances and uses thereof obtainable by the method for screening substances that inhibit binding between TAK1 and TAB1.
Transforming growth factor-xcex2 (TGF-xcex2) is a multilfunctional factor that controls various aspects of cell functions. As one such function, TGF-xcex2 is responsible for the repair and regeneration of tissues associated with various injuries (Border, W. A. and Noble, N. A., The New England Journal of Medicine (1994) 331, 1286-1292).
An abnormal production of TGF-xcex2 in chronic injuries can sometimes disturb balances in the repair and regeneration of tissues resulting in pathological fibrosis. As a pathological condition in which the balance of TGF-xcex2 production has been disturbed, hepatic fibrosis is known. It has been elucidated that TGF-xcex2 acts as a main causative agent of fibrosis of various organs such as the liver, by enhancing the production of extracellular matrix protein that can cause fibrosis, inhibiting the synthesis of proteolytic enzymes of extracellular matrix, and by inducing substances that inhibit proteolytic enzymes of extracellular matrix (Border, W. A. and Noble, N. A., The New England Journal of Medicine (1994) 331, 1286-1292).
Other known functions of TGF-xcex2 include the activity of inhibiting cellular growth (Moses, H. L. et al., Cell (1990) 63, 245-247), the activity of migrating monocytes (Wahl, S. M. et al., Proc. Natl. Acad. Sci. U.S.A. (1987) 84, 5788-5792), the activity of inducing biologically active substances (Wahl, S. M. et al., Proc. Natl. Acad. Sci. U.S.A. (1987) 84, 5788-5792), the activity of facilitating the deposition of amyloid xcex2 protein (Wyss-Coray, T. et al., Nature (1997) 389, 603-606), and the like.
TGF-xcex2 transduces its signals through heteromer complexes of type I and type II TGF-xcex2 receptors and transmembrane proteins containing the serine- and threonine-specific kinase domains at the side of cytoplasm (Wrana, J. L. et al., Nature (1994) 370, 341; Kingsley, D. M. et al., Genes Dev. (1994) 8, 133). However, much of the mechanism of signaling downward from the TGF-xcex2 receptor into the cell on the molecular level remains to be elucidated.
As a series of systems involved in the signal transduction of the TGF-xcex2 superfamily, mitogen-activated protein kinase (MAPK) is known.
The MAPK system is a conserved eukaryotic signaling system that converts signals of a receptor into various functions. The MAPK system contains three types of protein kinases, i.e. mitogen-activated protein kinase kinase kinase (MAPKKK), mitogen-activated protein kinase kinase (MAPKK), and mitogen-activated protein kinase (MAPK). MAPK is activated through phosphorylation by MAPKK. MAPKK is activated through phosphorylation by MAPKKK (Nishida, E. et al., Trends Biochem. Sci. (1993) 18, 128; Blumer, K. J. et al., Trends Biochem. Sci. (1993) 19, 236; David R. J. et al., Trends Biochem. Sci. (1993) 19, 470; Marchall, C. J. et al., Cell (1995) 80, 179).
TAK1 (TGF-xcex2-activated kinase 1), that is a member of the MAPKKK family that functions in the signaling system of biologically active substances and that belongs to the TGF-xcex2 superfamily, was identified by Yamaguchi, K. et. al. (Yamaguchi, K. et al., Science (1995) 270, 2008).
TAB1 (TAK1 binding protein 1), a protein involved in the signaling system of TGF-xcex2 that binds to and activates TAK1, was identified by Shibuya, H. et al. (Shibuya, H. et al., Science (1996) 272, 1179-1182).
Although TAB1 transduces the signal of TGF-xcex2 by binding to TAK1 and activating TAK1 kinase activity, no attempts have been made so far to search for substances that inhibit binding between TAK1 and TAB1 in order to suppress or activate signal transduction of TGF-xcex2 by focusing on the binding between TAK1 and TAB1.
The present invention is intended to provide a method for screening substances that inhibit binding between TAK1 and TAB1. The present invention is also intended to provide a method for screening substances that suppress or activate the signal transduction of TGF-xcex2. The present invention further is intended to provide substances that are obtainable by a method for screening substances that inhibit binding between TAK1 and TAB1.
Thus, the present invention provides (1) a method for screening substances that inhibit binding between a TAK1 polypeptide and a TAB1 polypeptide, which method comprises contacting the TAB1 polypeptide to the TAK1 polypeptide and a test sample and then detecting or determining the TAK1 polypeptide that is bound to the TAB1 polypeptide. Preferably, the TAB1 polypeptide is a TAB1 polypeptide that has been bound to a support. A preferred support is beads or a plate. In another preferred embodiment, the contact between a TAK1 polypeptide, a TAB1 polypeptide and a test sample is carried out in a homogeneous system.
The present invention also provides (2) a method for screening substances that inhibit binding between a TAK1 polypeptide and a TAB1 polypeptide, which method comprises contacting the TAK1 polypeptide to the TAB1 polypeptide and a test sample, and then detecting or determining the TAB1 polypeptide that is bound to the TAK1 polypeptide. Preferably, the TAK1 polypeptide is a TAK1 polypeptide that has been bound to a support. A preferred support is beads or a plate. In another preferred embodiment, the contact between a TAK1 polypeptide, a TAB1 polypeptide and a test sample is carried out in a homogeneous system.
The present invention also provides (3) a screening method described in the above (1) and (2), which method comprises using a TAB1 polypeptide having an amino acid sequence comprising Met at amino acid position 1 to Pro at amino acid position 504 of the amino acid sequence as set forth in SEQ ID NO: 2, or having an amino acid sequence modified by the substitution, deletion and/or addition of one or a plurality of amino acid residues of the amino acid sequence as set forth in SEQ ID NO: 2 and maintaining the biological activity of the TAB1 polypeptide; and/or
a TAK1 polypeptide having an amino acid sequence comprising Met at amino acid position 1 to Ser at amino acid position 579 of the amino acid sequence as set forth in SEQ ID NO: 4, or having an amino acid sequence modified by the substitution, deletion and/or addition of one or a plurality of amino acid residues of the amino acid sequence as set forth in SEQ ID NO: 4 and maintaining the biological activity of the TAK1 polypeptide.
The present invention also provides (4) a screening method described in the above (1) to (3), which comprises using a TAK1 polypeptide fused to another peptide or polypeptide and/or a TAB1 polypeptide fused to another peptide or polypeptide.
The present invention also provides (5) a method for screening substances that inhibit binding between a TAK1 polypeptide and a TAB1 polypeptide, which method comprises contacting the TAB1 polypeptide to the labeled TAK1 polypeptide and a test sample, and then detecting or determining the labeled TAK1 polypeptide that is bound to the TAB1 polypeptide. Preferably, the TAB1 polypeptide is a TAB1 polypeptide that has been bound to a support. A preferred support is beads or a plate. In another preferred embodiment, the contact between a TAK1 polypeptide, a TAB1 polypeptide and a test sample is carried out in a homogeneous system.
The present invention also provides (6) a method for screening substances that inhibit binding between a TAK1 polypeptide and a TAB1 polypeptide, which method comprises contacting the TAK1 polypeptide to the labeled TAB1 polypeptide and a test sample, and then detecting or determining the labeled TAB1 polypeptide that is bound to the TAK1 polypeptide. Preferably, the TAK1 polypeptide is a TAK1 polypeptide that has been bound to a support. A preferred support is beads or a plate. In another preferred embodiment, the contact between a TAK1 polypeptide, a TAB1 polypeptide and a test sample is carried out in a homogeneous system.
The present invention also provides (7) a screening method described in the above (5) and (6), which method comprises using a TAB1 polypeptide having an amino acid sequence comprising Met at amino acid position 1 to Pro at amino acid position 504 of the amino acid sequence as set forth in SEQ ID NO: 2 or having an amino acid sequence modified by the substitution, deletion and/or addition of one or a plurality of amino acid residues of the amino acid sequence as set forth in SEQ ID NO: 2 and maintaining the biological activity of the TAB1 polypeptide; and/or
a TAK1 polypeptide having an amino acid sequence comprising Met at amino acid position 1 to Ser at amino acid position 579 of the amino acid sequence as set forth in SEQ ID NO: 4 or having an amino acid sequence modified by the substitution, deletion and/or addition of one or a plurality of amino acid residues of the amino acid sequence as set forth in SEQ ID NO: 4 and maintaining the biological activity of the TAK1 polypeptide.
The present invention also provides (8) a screening method described in the above (5) to (7), which method comprises using a TAK1 polypeptide fused to another peptide or polypeptide and/or a TAB1 polypeptide fused to another peptide or polypeptide. Preferably, said labeled TAK1 polypeptide or said labeled TAB1 polypeptide is a TAK1 polypeptide or a TAB1 polypeptide that is labeled with a radioisotope, an enzyme or a fluorescent substance.
The present invention also provides (9) a method for screening substances that inhibit binding between a TAK1 polypeptide and a TAB1 polypeptide, which method comprises contacting the TAB1 polypeptide to the TAK1 polypeptide and a test sample, and then detecting or determining the TAK1 polypeptide that is bound to the TAB1 polypeptide by a primary antibody against the TAK1 polypeptide. Preferably, the TAB1 polypeptide is a TAB1 polypeptide that has been bound to a support. A preferred support is beads or a plate. Preferably, the primary antibody is a primary antibody that is labeled with a radioisotope or an enzyme. In another preferred embodiment, the contact between a TAK1 polypeptide, a TAB1 polypeptide and a test sample is carried out in a homogeneous system.
The present invention also provides (10) a method for screening substances that inhibit binding between a TAK1 polypeptide and a TAB1 polypeptide, which method comprises contacting the TAK1 polypeptide to the TAB1 polypeptide and a test sample, and then detecting or determining the TAB1 polypeptide that is bound to the TAK1 polypeptide by a primary antibody against the TAB1 polypeptide. Preferably, the TAK1 polypeptide is a TAK1 polypeptide that has been bound to a support. A preferred support is beads or a plate. Preferably, the primary antibody is a primary antibody that is labeled with a radioisotope, an enzyme or a fluorescent substance. In another preferred embodiment, the contact between a TAK1 polypeptide, a TAB1 polypeptide and a test sample is carried out in a homogeneous system.
The present invention also provides (11) a screening method described in the above (9) and (10), which method comprises using a TAB1 polypeptide having an amino acid sequence comprising Met at amino acid position 1 to Pro at amino acid position 504 of the amino acid sequence as set forth in SEQ ID NO: 2 or having an amino acid sequence modified by the substitution, deletion and/or addition of one or a plurality of amino acid residues of the amino acid sequence as set forth in SEQ ID NO: 2 and maintaining the biological activity of the TAB1 polypeptide.
a TAK1 polypeptide having an amino acid sequence comprising Met at amino acid position 1 to Ser at amino acid position 579 of the amino acid sequence as set forth in SEQ ID NO: 4 or having an amino acid sequence modified by the substitution, deletion and/or addition of one or a plurality of amino acid residues of the amino acid sequence as set forth in SEQ ID NO: 4 and maintaining the biological activity of the TAK1 polypeptide.
The present invention also provides (12) a screening method described in the above (9) to (11), which method comprises using a TAK1 polypeptide fused to another peptide or polypeptide and/or a TAB1 polypeptide fused to another peptide or polypeptide.
The present invention also provides (13) a method for screening substances that inhibit binding between a TAK1 polypeptide and a TAB1 polypeptide, which method comprises contacting the TAB1 polypeptide or the TAB1 polypeptide fused to another peptide or polypeptide to the TAK1 polypeptide fused to another peptide or polypeptide and a test sample, and then detecting or determining the TAK1 polypeptide fused to another peptide or polypeptide said TAK1 polypeptide being bound to the TAB1 polypeptide or the TAB1 polypeptide fused to another peptide or polypeptide by a primary antibody against the other peptide or polypeptide. Preferably, the TAB1 polypeptide or the TAB1 polypeptide fused to another peptide or polypeptide is a TAB1 polypeptide or a TAB1 polypeptide fused to another peptide or polypeptide, that has been bound to a support. A preferred support is beads or a plate. Preferably, the primary antibody is a primary antibody that is labeled with a radioisotope or an enzyme. In another preferred embodiment, the contact between a TAK1 polypeptide, a TAB1 polypeptide and a test sample is carried out in a homogeneous system.
The present invention also provides (14) a method for screening substances that inhibit binding between a TAK1 polypeptide and a TAB1 polypeptide, which method comprises contacting the TAK1 polypeptide or the TAK1 polypeptide fused to another peptide or polypeptide to the TAB1 polypeptide fused to another peptide or polypeptide and a test sample, and then detecting or determining the TAB1 polypeptide fused to another peptide or polypeptide said TAB1 polypeptide being bound to the TAK1 polypeptide or the TAK1 polypeptide fused to another peptide or polypeptide, by a primary antibody against the other peptide or polypeptide. Preferably, the TAK1 polypeptide or the TAK1 polypeptide fused to another peptide or polypeptide is a TAK1 polypeptide or a TAK1 polypeptide fused to another peptide or polypeptide, that has been bound to a support. A preferred support is beads or a plate. Preferably, the primary antibody is a primary antibody that is labeled with a radioisotope, an enzyme a fluorescent substance. In another preferred embodiment, the contact between a TAK1 polypeptide, a TAB1 polypeptide and a test sample is carried out in a homogeneous system.
The present invention also provides (15) a screening method described in the above (13) and (14), which method comprises using a TAB1 polypeptide having an amino acid sequence comprising Met at amino acid position 1 to Pro at amino acid position 504 of the amino acid sequence as set forth in SEQ ID NO: 2 or having an amino acid sequence modified by the substitution, deletion and/or addition of one or a plurality of amino acid residues of the amino acid sequence as set forth in SEQ ID NO: 2 and maintaining the biological activity of the TAB1 polypeptide; and/or
a TAK1 polypeptide having an amino acid sequence comprising Met at amino acid position 1 to Ser at amino acid position 579 of the amino acid sequence as set forth in SEQ ID NO: 4 or having an amino acid sequence modified by the substitution, deletion and/or addition of one or a plurality of amino acid residues of the amino acid sequence as set forth in SEQ ID NO: 4 and maintaining the biological activity of the TAK1 polypeptide.
The present invention also provides (16) a method for screening substances that inhibit binding between a TAK1 polypeptide and a TAB1 polypeptide, which method comprises contacting the TAB1 polypeptide to the TAK1 polypeptide and a test sample, and then detecting or determining the TAK1 polypeptide that is bound to the TAB1 polypeptide by a primary antibody against the TAK1 polypeptide and a secondary antibody against the primary antibody. Preferably, the TAB1 polypeptide is a TAB1 polypeptide that has been bound to a support. A preferred support is beads or a plate. Preferably, the secondary antibody is a secondary antibody that is labeled with a radioisotope, an enzyme or a fluorescent substance. In another preferred embodiment, the contact between a TAK1 polypeptide, a TAB1 polypeptide and a test sample is carried out in a homogeneous system.
The present invention also provides (17) a method for screening substances that inhibit binding between a TAK1 polypeptide and a TAB1 polypeptide, which method comprises contacting the TAK1 polypeptide to the TAB1 polypeptide and a test sample, and then detecting or determining the TAB1 polypeptide that is bound to the TAK1 polypeptide by a primary antibody against the TAB1 polypeptide and a secondary antibody against the primary antibody. Preferably, the TAK1 polypeptide is a TAK1 polypeptide that has been bound to a support. A preferred support is beads or a plate. Preferably, the secondary antibody is a secondary antibody that is labeled with a radioisotope, an enzyme or a fluorescent substance. In another preferred embodiment, the contact between a TAK1 polypeptide, a TAB1 polypeptide and a test sample is carried out in a homogeneous system.
The present invention also provides (18) a screening method described in the above (16) and (17), which method comprises using a TAB1 polypeptide having an amino acid sequence comprising Met at amino acid position 1 to Pro at amino acid position 504 of the amino acid sequence as set forth in SEQ ID NO: 2 or having an amino acid sequence modified by the substitution, deletion and/or addition of one or a plurality of amino acid residues of the amino acid sequence as set forth in SEQ ID NO: 2 and maintaining the biological activity of the TAB1 polypeptide; and/or
a TAK1 polypeptide having an amino acid sequence comprising Met at amino acid position 1 to Ser at amino acid position 579 of the amino acid sequence as set forth in SEQ ID NO: 4 or having an amino acid sequence modified by the substitution, deletion and/or addition of one or a plurality of amino acid residues of the amino acid sequence as set forth in SEQ ID NO: 4 and maintaining the biological activity of the TAK1 polypeptide.
The present invention also provides (19) a screening method described in the above (16) to (18), which method comprises using a TAK1 polypeptide fused to another peptide or polypeptide and/or a TAB1 polypeptide fused to another peptide or polypeptide.
The present invention also provides (20) a method for screening substances that inhibit binding between a TAK1 polypeptide and a TAB1 polypeptide, which method comprises contacting the TAB1 polypeptide or the TAB1 polypeptide fused to another peptide or polypeptide to the TAK1 polypeptide fused to another peptide or polypeptide and a test sample, and then detecting or determining the TAK1 polypeptide fused to another peptide or polypeptide said TAK1 polypeptide being bound to the TAB1 polypeptide or the TAB1 polypeptide fused to another peptide or polypeptide, by a primary antibody against the other peptide or polypeptide and a secondary antibody against the primary antibody. Preferably, the TAB1 polypeptide or the TAB1 polypeptide fused to another peptide or polypeptide is a TAB1 polypeptide or the TAB1 polypeptide fused to another peptide or polypeptide, that has been bound to a support. A preferred support is beads or a plate. Preferably, the secondary antibody is a primary antibody that is labeled with a radioisotope, an enzyme or a fluorescent substance. In another preferred embodiment, the contact between a TAK1 polypeptide, a TAB1 polypeptide and a test sample is carried out in a homogeneous system.
The present invention also provides (21) a method for screening substances that inhibit binding between a TAK1 polypeptide and a TAB1 polypeptide, which method comprises contacting the TAK1 polypeptide or the TAK1 polypeptide fused to another peptide or polypeptide to the TAB1 polypeptide fused to another peptide or polypeptide and a test sample, and then detecting or determining the TAB1 polypeptide fused to another peptide or polypeptide said TAB1 polypeptide being bound to the TAK1 polypeptide or the TAK1 polypeptide fused to another peptide or polypeptide, by a primary antibody against the other peptide or polypeptide and a secondary antibody against the primary antibody. Preferably, the TAK1 polypeptide or the TAK1 polypeptide fused to another peptide or polypeptide is a TAK1 polypeptide or the TAK1 polypeptide fused to another peptide or polypeptide, that has been bound to a support. A preferred support is beads or a plate. Preferably, the secondary antibody is a secondary antibody that is labeled with a radioisotope, an enzyme a fluorescent substance. In another preferred embodiment, the contact between a TAK1 polypeptide, a TAB1 polypeptide and a test sample is carried out in a homogeneous system.
The present invention also provides (22) a screening method described in the above (20) and (21), which method comprises using a TAB1 polypeptide having an amino acid sequence comprising Met at amino acid position 1 to Pro at amino acid position 504 of the amino acid sequence as set forth in SEQ ID NO: 2 or having an amino acid sequence modified by the substitution, deletion and/or addition of one or a plurality of amino acid residues of the amino acid sequence as set forth in SEQ ID NO: 2 and maintaining the biological activity of the TAB1 polypeptide; and/or
a TAK1 polypeptide having an amino acid sequence comprising Met at amino acid position 1 to Ser at amino acid position 579 of the amino acid sequence as set forth in SEQ ID NO: 4 or having an amino acid sequence modified by the substitution, deletion and/or addition of one or a plurality of amino acid residues of the amino acid sequence as set forth in SEQ ID NO: 4 and maintaining the biological activity of the TAK1 polypeptide.
The present invention also provides a kit for conducting the screening method as set forth in any of the above (1) to (22).
The present invention also provides a substance that is obtainable by the screening method as set forth in any of the above (1) to (22).
The present invention also provides a substance that inhibits binding between a TAK1 polypeptide and a TAB1 polypeptide obtainable by the screening method as set forth in any of the above (1) to (22).
The present invention also provides an inhibitor of signal transduction of TGF-xcex2, said inhibitor comprising a substance that inhibits binding between a TAK1 polypeptide and a TAB1 polypeptide, obtainable by the screening method as set forth in any of the above (1) to (22).
The present invention also provides an activator of signal transduction of TGF-xcex2, said activator comprising a substance that inhibits binding between a TAK1 polypeptide and a TAB1 polypeptide, obtainable by the screening method as set forth in any of the above (1) to (22).
The present invention also provides a suppressor of the enhancement of extracellular matrix protein production, said suppressor comprising, as an active ingredient, a substance that inhibits binding between a TAK1 polypeptide and a TAB1 polypeptide, obtainable by the screening method as set forth in any of the above (1) to (22).
The present invention also provides an activator of the enhancement of extracellular matrix protein production, said activator comprising as an active ingredient, a substance that inhibits binding between a TAK1 polypeptide and a TAB1 polypeptide, obtainable by the screening method as set forth in any of the above (1) to (22).
The present invention also provides a suppressor of the inhibition of cellular growth, said suppressor comprising, as an active ingredient, a substance that inhibits binding between a TAK1 polypeptide and a TAB1 polypeptide, obtainable by the screening method as set forth in any of the above (1) to (22).
The present invention also provides an activator of the inhibition of cellular growth, said activator comprising, as an active ingredient, a substance that inhibits binding between a TAK1 polypeptide and a TAB1 polypeptide, obtainable by the screening method as set forth in any of the above (1) to (22).
The present invention also provides a suppressor of monocyte migration, said suppressor comprising, as an active ingredient, a substance that inhibits binding between a TAK1 polypeptide and a TAB1 polypeptide, obtainable by the screening method as set forth in any of the above (1) to (22).
The present invention also provides an activator of monocyte migration, said activator comprising, as an active ingredient, a substance that inhibits binding between a TAK1 polypeptide and a TAB1 polypeptide, obtainable by the screening method as set forth in any of the above (1) to (22).
The present invention also provides a suppressor of the induction of a biologically active substance, said suppressor comprising, as an active ingredient, a substance that inhibits binding between a TAK1 polypeptide and a TAB1 polypeptide, obtainable by the screening method as set forth in any of the above (1) to (22).
The present invention also provides an activator of a biologically active substance, said activator comprising, as an active ingredient, a substance that inhibits binding between a TAK1 polypeptide and a TAB1 polypeptide, obtainable by the screening method as set forth in any of the above (1) to (22).
The present invention also provides a suppressor of an immunosuppressive action, said suppressor comprising, as an active ingredient, a substance that inhibits binding between a TAK1 polypeptide and a TAB1 polypeptide, obtainable by the screening method as set forth in any of the above (1) to (22).
The present invention also provides an activator of an immunosuppressive action, said activator comprising, as an active ingredient, a substance that inhibits binding between a TAK1 polypeptide and a TAB1 polypeptide, obtainable by the screening method as set forth in any of the above (1) to (22).
The present invention also provides a suppressor of the deposition of amyloid xcex2 protein, said suppressor comprising, as an active ingredient, a substance that inhibits binding between a TAK1 polypeptide and a TAB1 polypeptide, obtainable by the screening method as set forth in any of the above (1) to (22).
The present invention also provides an activator of the deposition of amyloid xcex2 protein, said activator comprising, as an active ingredient, a substance that inhibits binding between a TAK1 polypeptide and a TAB1 polypeptide, obtainable by the screening method as set forth in any of the above (1) to (22).